Recent advances in molecular biology have led to an appreciation of the central role of signal transduction pathways in biological processes. These pathways comprise a central means by which individual cells in a multicellular organism communicate, thereby coordinating biological processes. See Springer, Cell 76:301-314 (1994), Table I, for a model. One branch of signal transduction pathways, defined by the intracellular participation of guanine nucleotide binding proteins (G-proteins), affects a broad range of biological processes.
Lewin, GENES V 319-348 (1994) generally discusses G-protein signal transduction pathways which involve, at a minimum, the following components: an extracellular signal (e.g., neurotransmitters, peptide hormones, organic molecules, light, or odorants), a signal-recognizing receptor [G-protein-coupled receptor, reviewed in Probst et al., DNA and Cell Biology 11:1-20 (1992) and also known as GPR or GPCR], and an intracellular, heterotrimeric GTP-binding protein, or G protein. In particular, these pathways have attracted interest because of their role in regulating white blood cell or leukocyte trafficking.
Leukocytes comprise a group of mobile blood cell types including granulocytes (i.e., neutrophils, basophils, and eosinophils), lymphocytes, and monocytes. When mobilized and activated, these cells are primarily involved in the body's defense against foreign matter. This task is complicated by the diversity of normal and pathological processes in which leukocytes participate. For example, leukocytes function in the normal inflammatory response to infection. Leukocytes are also involved in a variety of pathological inflammations. For a summary, see Schall et al., Curr. Opin. Immunol. 6:865-873 (1994). Moreover, each of these processes can involve unique contributions, in degree, kind, and duration, from each of the leukocyte cell types.
In studying these immune reactions, researchers initially concentrated on the signals acting upon leukocytes, reasoning that a signal would be required to elicit any form of response. Murphy, Ann. Rev. Immunol. 12:593-633 (1994) has reviewed members of an important group of leukocyte signals, the peptide signals. One type of peptide signal comprises the chemokines (chemoattractant cytokines), termed intercrines in Oppenheim et al., Ann. Rev. Immunol. 9:617-648 (1991). In addition to Oppenheim et al., Baggiolini et al., Advances in Immunol. 55:97-179 (1994), documents the growing number of chemokines that have been identified and subjected to genetic and biochemical analyses.
Comparisons of the amino acid sequences of the known chemokines have led to a classification scheme which divides chemokines into two groups: the α group characterized by a single amino acid separating the first two cysteines (CXC; N-terminus as referent), and the β group, where these cysteines are adjacent (CC). See Baggiolini et al., supra. Correlations have been found between the chemokines and the particular leukocyte cell types responding to those signals. Schall et al., supra, has reported that the CXC chemokines generally affect neutrophils; the CC chemokines tend to affect monocytes, lymphocytes, basophils and eosinophils. For example, Baggiolini et al., supra, recited that RANTES, a CC chemokine, functions as a chemoattractant for monocytes, lymphocytes (i.e., memory T cells), basophils, and eosinophils, but not for neutrophils, while inducing the release of histamine from basophils.
Chemokines were recently shown by Cocchi et. al., Science, 270: 1811-1815 (1995) to be suppressors of HIV proliferation. Cocchi et al. (supra) demonstrated that RANTES, MIP-1α, and MIP-1β suppressed HIV-1, HIV-2 and SIV infection of a CD4+ cell line designated PM1 and of primary human peripheral blood mononuclear cells.
Recently, however, attention has turned to the cellular receptors that bind the chemokines, because the extracellular chemokines seem to contact cells indiscriminately, and therefore lack the specificity needed to regulate the individual leukocyte cell types.
Murphy (supra) reported that the GPCR superfamily of receptors includes the chemokine receptor family. The typical chemokine receptor structure includes an extracellular chemokine-binding domain located near the N-terminus, followed by seven spaced regions of predominantly hydrophobic amino acids capable of forming membrane-spanning α-helices. Between each of the α-helical domains are hydrophilic domains localized, alternately, in the intra- or extra-cellular spaces. These features impart a serpentine conformation to the membrane-embedded chemokine receptor. The third intracellular loop typically interacts with G-proteins. In addition, Murphy (supra) noted that the intracellular carboxyl terminus is also capable of interacting with G-proteins.
The first chemokine receptors to be analyzed by molecular cloning techniques were the two neutrophil receptors for human IL8, a CXC chemokine. Holmes et al., Science 253:178-1280 (1991) and Murphy et al., Science 253:1280-1283 (1991), reported the cloning of these two receptors for IL8. Lee et al., J. Biol. Chem. 267:16283-16287 (1992), analyzed the cDNAs encoding these receptors and found 77% amino acid identity between the encoded receptors, with each receptor exhibiting features of the G protein coupled receptor family. One of these receptors is specific for IL-8, while the other binds and signals in response to IL-8, gro/MGSA, and NAP-2. Genetic manipulation of the genes encoding IL-8 receptors has contributed to our understanding of the biological roles occupied by these receptors. For example, Cacalano et al., Science 265:682-684 (1994) reported that deletion of the IL-8 receptor homolog in the mouse resulted in a pleiotropic phenotype involving lymphadenopathy and splenomegaly. In addition, a study of missense mutations described in Leong et al., J. Biol. Chem. 269:19343-19348 (1994) revealed amino acids in the IL-8 receptor that were critical for IL-8 binding. Domain swapping experiments discussed in Murphy (supra) implicated the amino terminal extracellular domain as a determinant of binding specificity.
Several receptors for CC chemokines have also been identified and cloned. CCCKR1 binds both MIP-1αand RANTES and causes intracellular calcium ion flux in response to both ligands. Charo et al., Proc Natl. Acad. Sci. (USA) 91:2752-2756 (1994) reported that another CC chemokine receptor, MCP-R1 (CCCKR2), is encoded by a single gene that produces two splice variants which differ in their carboxyl terminal domains. This receptor binds and responds to MCP-3 in addition to MCP-1.
A promiscuous receptor that binds both CXC and CC chemokines has also been identified. This receptor was originally identified on red blood cells and Horuk et al., Science 261:1182-1184 (1993) reports that it binds IL-8, NAP-2, GROα, RANTES, and MCP-1. The erythrocyte chemokine receptor shares about 25% identity with other chemokine receptors and may help to regulate circulating levels of chemokines or aid in the presentation of chemokines to their targets. In addition to binding chemokines, the erythrocyte chemokine receptor has also been shown to be the receptor for plasmodium vivax, a major cause of malaria (id.) Another G-protein coupled receptor which is closely related to chemokine receptors, the platelet activating factor receptor, has also been shown to be the receptor for a human pathogen, the bacterium Streptococcus pneumoniae [Cundell et al., Nature 377:435-438 (1995)].
In addition to the mammalian chemokine receptors, two viral chemokine receptor homologs have been identified. Ahuja et al., J. Biol. Chem. 268:20691-20694 (1993) describes a gene product from Herpesvirus saimiri that shares about 30% identity with the IL-8 receptors and binds CXC chemokines. Neote et al., Cell, 72:415-425 (1993) reports that human cytomegalovirus contains a gene encoding a receptor sharing about 30% identity with the CC chemokine receptors which binds MIP-1α, MIP-1β, MCP-1, and RANTES. These viral receptors may affect the normal role of chemokines and provide a selective pathological advantage for the virus.
Because of the broad diversity of chemokines and their activities, there are numerous receptors for the chemokines. The receptors which have been characterized represent only a fraction of the total complement of chemokine receptors. There thus remains a need in the art for the identification of additional chemokine receptors. The availability of these novel receptors will provide tools for the development of therapeutic modulators of chemokine or chemokine receptor function. It is contemplated by the present invention that such modulators are useful as therapeutics for the treatment of atherosclerosis, rheumatoid arthritis, tumor growth suppression, asthma, viral infections, and other inflammatory conditions. Alternatively, fragments or variants of the chemokine receptors, or antibodies recognizing those receptors, are contemplated as therapeutics.